Novel,Monomeric Cyanine Dyes as Reporters for DNA Helicase Activity

The dimeric cyanine dyes, YOYO-1 and TOTO-1, are widely used as DNA probes because of their excellent fluorescent properties. They have a higher fluorescence quantum yield than ethidium homodimer, DAPI and Hoechst dyes and bind to double-stranded DNA with high affinity. However, these dyes are limited by heterogeneous staining at high dye loading, photocleavage of DNA under extended illumination, nicking of DNA, and inhibition of the activity of DNA binding enzymes. To overcome these limitations, seven novel cyanine dyes (Cyan-2, DC-21, DM, DM-1, DMB-2OH, SH-0367, SH1015-OH) were synthesized and tested for fluorescence emission, resistance to displacement by Mg²⁺, and the ability to function as reporters for DNA unwinding. Results show that Cyan-2, DM-1, SH-0367 and SH1015-OH formed highly fluorescent complexes with dsDNA. Of these, only Cyan-2 and DM-1 exhibited a large fluorescence enhancement in buffers, and were resistant to displacement by Mg²⁺. The potential of these two dyes to function as reporter molecules was evaluated using continuous fluorescence, DNA helicase assays. The rate of DNA unwinding was not significantly affected by either of these two dyes. Therefore, Cyan-2 and DM-1 form the basis for the synthesis of novel cyanine dyes with the potential to overcome the limitations of YOYO-1 and TOTO-1.     

Mono and Trimethine Cyanines Cyan 40 and Cyan 2 as Probes for Highly Selective Fluorescent Detection of Non-canonical DNA Structures

Two of earlier reported dsDNA sensitive cyanine dyes??monomethine Cyan 40 and meso-substituted trimethine Cyan 2 were studied for their ability to interact with non-canonical DNA conformations. These dyes were characterized by spectral-luminescent methods in the presence of G-quadruplex, triplex and dsDNA motifs. We have demonstrated that Cyan 2 binds strongly and preferentially to triple- and quadruple-stranded DNA forms that results in a strong enhancement of the dye fluorescence, as compared to dsDNA, while Cyan 40 form fluorescent complexes preferentially only with the triplex form. Highly fluorescent complexes of Cyan 2 with DNA triplexes and G-quadruplexes and Cyan 40 with DNA triplexes are very stable and do not dissociate during gel electrophoresis, leading to preferential staining of the above DNA forms in gels. The data presented point to the intercalation mechanism of the Cyan 2 binding to G4-DNA, while the complexes of Cyan 40 and Cyan 2 with triplex DNA are believed to be formed via groove binding mode. The Cyan dyes can provide a highly sensitive method for detection and quantification of non-canonical structures in genome.     

Direct Observation Method of Individual Single-Stranded DNA Molecules Using Fluorescent Replication Protein A

Direct observation studies of single molecules have revealed molecular behaviors usually hidden in the ensemble and time-averaging of bulk experiments. Direct single DNA molecule analysis of DNA metabolism reactions such as DNA replication, repair, and recombination is necessary to fully understand these essential processes. Intercalation of fluorescent dyes such as YOYO-1 and SYTOX Orange has been the standard method for observing single molecules of double-stranded DNA (dsDNA), but effective fluorescent dyes for observing single molecules of single-stranded DNA (ssDNA) have not been found. To facilitate direct single-molecule observations of DNA metabolism reactions, it is necessary to establish methods for discriminating ssDNA and dsDNA. To observe ssDNA directly, we prepared a fusion protein consisting of the 70 kDa DNA-binding domain of replication protein A and enhanced yellow fluorescent protein (RPA-YFP). This fusion protein had ssDNA-binding activity. In our experiments, dsDNA was stained by SYTOX Orange and ssDNA by RPA-YFP, and we succeeded in staining ssDNA and dsDNA by using RPA-YFP and SYTOX Orange simultaneously.     

Xe1v离子激光感生荧光用于医用X射线ZnCdS:Ag荧光屏的性能研究

本文叙述了利用同轴结构Xe1v离子激光器的UVλ=365nm激光作为激发光源的激光感生荧光(LIF)实验系统,对医用X射线ZnCdS:Ag荧光屏的发光性能进行了研究.LIF方法能方便而高灵敏地同时测得荧光光谱与寿命,和传统的紫外灯激发及X射线激发的结果一致,可以完全避免X射线对人体的有害辐射.     

SYBR Green I: Fluorescence Properties and Interaction with DNA

In this study, we have investigated the fluorescence properties of SYBR Green I (SG) dye and its interaction with double-stranded DNA (dsDNA). SG/dsDNA complexes were studied using various spectroscopic techniques, including fluorescence resonance energy transfer and time-resolved fluorescence techniques. It is shown that SG quenching in the free state has an intrinsic intramolecular origin; thus, the observed >1,000-fold SG fluorescence enhancement in complex with DNA can be explained by a dampening of its intra-molecular motions. Analysis of the obtained SG/DNA binding isotherms in solutions of different ionic strength and of SG/DNA association in the presence of a DNA minor groove binder, Hoechst 33258, revealed multiple modes of interaction of SG inner groups with DNA. In addition to interaction within the DNA minor groove, both intercalation between base pairs and stabilization of the electrostatic SG/DNA complex contributed to increased SG affinity to double-stranded DNA. We show that both fluorescence and the excited state lifetime of SG dramatically increase in viscous solvents, demonstrating an approximate 200-fold enhancement in 100?% glycerol, compared to water, which also makes SG a prospective fluorescent viscosity probe. A proposed structural model of the SG/DNA complex is compared and discussed with results recently reported for the closely related PicoGreen chromophore.     

Fluorescence Characteristics of Variously Charged Asymmetric Monomethine Cyanine Dyes in the Presence of Nucleic Acids

Three asymmetric monomethine cyanine dyes bearing one, two, and three positive charges have been synthesized, and their absorption and fluorescence characteristics in the presence of nucleic acids were studied. The maxima of their longest wavelength absorption band lie between 500 and 520 nm. The dyes do not show fluorescence of their own in TE buffer (pH = 7.5), but become strongly fluorescent (Q_F = 0.2–0.6) on binding to double-stranded DNA. The fluorescence maxima of the investigated dye-dsDNA complexes are in the region of 530–550 nm. The influence of the dye/DNA ratio on both the position and intensity of the fluorescence maxima of the complexes is investigated.     

Fluorescence Study of DNA–Dye Complexes Using One-Photon and Two-Photon Picosecond Excitation

Preliminary results of investigation of one-photon- and two-photon-induced fluorescence of acridine orange (AO), epirubicin (ER), hypericin (HYP), and ethidium bromide (EB) in complexes with DNA are presented. A spectrofluorometer based on a picosecond Nd:YAG laser was used for investigations of two-photon (1064-nm, 1-mJ, 40-ps) and one-photon (532- and 355-nm) dye excitation. The spectra of two-photon-induced fluorescence of dyes and their complexes with DNA as well as the kinetics of dyes' fluorescence intensification during their interactions with DNA in dependence on the biomacromolecule concentration were obtained. The intensities of AO, HYP, and EB fluorescence were increased 2.4, 3.2, and 8 times, respectively, after binding with DNA at two-photon excitation, while at one-photon excitation the corresponding values were 2.5, 3.7, and 10 times. The difference in fluorescence enhancement during DNA–dye complex formation at linear and nonlinear excitation may possibly be associated with the fact that the cross sections of one-photon and two-photon absorption, in general, change unequally during the binding of dyes to organic molecules and bathocromic shift of the electronic transitions. It was shown that the peak of AO fluorescence shifted to a longer wavelength on 10 nm after two-photon excitation at 1064 nm in comparison with one-photon excitation at 532 nm.     

He-Ne激光对小麦幼苗增强UV-B辐射损伤修复的影响

采用He-Ne激光(5mW/mm²)来辐照处理经增强UV-B(10.8kJ·m^-2·d^-1)辐射损伤的小麦幼苗,通过荧光光谱测定其中双链DNA(dsDNA)的含量,分析研究了He-Ne激光对小麦DNAUV-B损伤的切除修复的影响和机制,以探明激光对UV-B损伤的修复途径及机制.结果表明:He-Ne激光能明显增强UV-B辐射处理后小麦种子的萌发力;小麦对UV-B辐射损伤具有一定的切除修复能力,切除修复的高峰期发生在UV-B辐射后4～6h内;He-Ne激光主要通过促进小麦的切除修复途径影响小麦对UV-B损伤的修复,而且能增强小麦的切除修复能力,其促进作用在修复高峰期(5h)表现尤为明显.     

Laser Tunable Source for the Red and Near UV Regions of the Spectrum

The spectral and lasing characteristics of a number of dyes for the red and near IR portions of the lasing spectrum in laser and lamp pumpings are investigated. Smooth tuning of a pulse-periodic lamp-pumped laser in the 660–720 nm range and generation of its second harmonic in the 330–360 nm region were obtained. As a result, the radiation range of the earlier developed tunable laser source has been extended in the red and near UV regions of the spectrum. The use of mixtures of dyes and proton-donating additives in working solutions has substantially improved the energy characteristics of this source.     

High-resolution nuclear track mapping in detailed cellular histology using CR-39 with the contact microscopy technique

In radiation cancer therapies using energetic charged particles such as proton/heavy-ion therapy and boron neutron capture therapy (BNCT), studies on radiation-induced biological response at cellular level are important because the radiation damage from energetic charged particles is highly localized along their paths and the radiation sensitivity is quite different in each cellular organelle. In such studies the information on the position of charged particle impact in biological cells is necessary. A novel method for high-resolution nuclear track mapping in detailed cellular histology has been developed. In this technique, biological specimens mounted on CR-39 plates are exposed to energetic charged particles. The irradiated samples are exposed to UV, and then etched for a short time. Both etch pits of nuclear tracks and relief for transmission UV image of the specimen can be observed on the CR-39 surface with an atomic force microscope (AFM) at about 100 nm resolution.     

Study on the Interaction of the CpG Alternating DNA with CdTe Quantum Dots

A novel sensitive method for detection of DNA methylation was developed with thioglycollic acid (TGA)-capped CdTe quantum dots (QDs) as fluorescence probes. Recognition of methylated DNA sites would be useful strategy due to the important roles of methylation in disease occurrence and developmental processes. DNA methylation occurs most often at cytosine-guanine sites (CpG dinucleotides) of gene promoters. The QDs significantly interacted with hybridized unmethylated and methylated DNA. The interaction of CpG rich methylated and unmethylated DNA hybrid with quantum dots as an optical probe has been investigated by fluorescence spectroscopy and electrophoresis assay. The fluorescence intensity of QDs was highly dependent to unmethylated and methylated DNA. Specific site of CpG islands of Adenomatous polyposis coli (APC), a well-studied tumor suppressor gene, was used as the detection target. Under optimum conditions, upon the addition of unmethylated dsDNA, the fluorescence intensity increased in linear range from 1.0?×?10^??10 to 1.0?×?10^??6M with detection limit of 6.2?×?10^??11 M and on the other hand, the intensity of QDs showed no changes with addition of methylated dsDNA. We also demonstrated that the unmethylated and methylated DNA and QDs complexes showed different mobility in electrophoresis assay. This easy and reliable method could distinguish between methylated and unmethylated DNA sequences.     

Two-Color Two-Photon Fluorescence Laser Scanning Microscopy

We present the first realization of a Two-Color Two-Photon Laser-Scanning Microscope (2c2pLSM) and UV fluorescence images of cells acquired with this technique. Fluorescence is induced by two-color two-photon absorption using the fundamental and the second harmonic of a Ti:Sa femtosecond laser. Simultaneous absorption of an 800 nm photon and a 400 nm photon energetically corresponds to one-photon absorption at 266 nm. This technique for Laser-Scanning Microscopy extends the excitation wavelength range of a Ti:Sa powered fluorescence microscope to the UV. In addition to the known advantages of multi-photon microscopy like intrinsic 3D resolution, reduced photo damage and high penetration depth 2c2pLSM offers the possibility of using standard high numeric aperture objectives for UV fluorescence imaging. The effective excitation wavelength of 266 nm corresponds especially well to the excitation spectrum of tryptophan. Hence, it is an ideal tool for label free fluorescence studies and imaging of intrinsic protein fluorescence which originates mainly from tryptophan. Thus a very sensitive natural lifetime probe can be used for monitoring protein reactions or changes in conformation. First measurements of living MIN-6 cells reveal differences between the UV fluorescence lifetimes of the nucleus and cytoplasm. The significance of this method was further demonstrated by monitoring the binding of biotin to avidin.     

The kinetics of force-dependent hybridization and strand-peeling of short DNA fragments

Deoxyribonucleic acid (DNA) carries the genetic information in all living organisms. It consists of two interwound single-stranded (ss) strands, forming a double-stranded (ds) DNA with a right-handed double-helical conformation. The two strands are held together by highly specific basepairing interactions and are further stabilized by stacking between adjacent basepairs. A transition from a dsDNA to two separated ssDNA is called melting and the reverse transition is called hybridization. Applying a tensile force to a dsDNA can result in a particular type of DNA melting, during which one ssDNA strand is peeled away from the other. In this work, we studied the kinetics of strand-peeling and hybridization of short DNA under tensile forces. Our results show that the force-dependent strand-peeling and hybridization can be described with a simple two-state model. Importantly, detailed analysis of the force-dependent transition rates revealed that the transition state consists of several basepairs dsDNA.     

Structure and Dynamics of dsDNA in Cell-like Environments

Deoxyribonucleic acid (DNA) is a fundamental biomolecule for correct cellular functioning and regulation of biological processes. DNA’s structure is dynamic and has the ability to adopt a variety of structural conformations in addition to its most widely known double-stranded DNA (dsDNA) helix structure. Stability and structural dynamics of dsDNA play an important role in molecular biology. In vivo, DNA molecules are folded in a tightly confined space, such as a cell chamber or a channel, and are highly dense in solution; their conformational properties are restricted, which affects their thermodynamics and mechanical properties. There are also many technical medical purposes for which DNA is placed in a confined space, such as gene therapy, DNA encapsulation, DNA mapping, etc. Physiological conditions and the nature of confined spaces have a significant influence on the opening or denaturation of DNA base pairs. In this review, we summarize the progress of research on the stability and dynamics of dsDNA in cell-like environments and discuss current challenges and future directions. We include studies on various thermal and mechanical properties of dsDNA in ionic solutions, molecular crowded environments, and confined spaces. By providing a better understanding of melting and unzipping of dsDNA in different environments, this review provides valuable guidelines for predicting DNA thermodynamic quantities and for designing DNA/RNA nanostructures.     

Excited state intramolecular proton transfer in amino 2-(2′-hydroxyphenyl)benzazole derivatives: Effects of the solvent and the amino group position

The excited-state intramolecular proton transfer (ESIPT) mechanism in six amino 2-(2′-hydroxyphenyl)benzazole derivatives were investigated in different solvents by means of UV-vis absorption and steady-state fluorescence. The amino benzazoles are fluorescent in the blue-orange region under UV radiation. Changes in the absorption, emission and excitation spectra were analyzed and correlated to the position of the amino group and the solvent polarity. The equilibrium between the conformers in solution in the ground state, confirmed by the solvatochromic effect, reflects the dual fluorescence emission presented by these dyes.     

Tb3+荧光离子探针检测三链DNA的形成

Tb^3 本身具有荧光,Tb^3 和DNA结合后仍具有荧光,荧光强度不仅与DNA中碱基的种类有关,而且还与DNA是单链、双链还是三链有关。文章用Tb^3 离子作为荧光探针,检测了三链DNA的形成。实验结果表明：polydA和Tb^3 结合后的荧光强度大于polyd T和Tb^3 结合后的荧光强度。说明荧光强度和碱基的种类有关。实验结果还显示Tb^3 在与三链DNA作用后的荧光光谱的谱峰位置与单链及双链DNA的荧光光谱的谱峰位置基本相同,但是其强度差异明显。Tb^3 与单链DNA作用后的荧光强度最大,三链次之,Tb^3 与双链DNA作用后的荧光强度最弱。通过测定荧光强度的变化研究了三链DNA的形成和pH、金属离子对形成三链DNA的影响。pH中性和高价阳离子的存在有利于三链DNA的形成。     

Spectroscopic study on the interaction of eugenol with salmon sperm DNA in vitro

Fluorescence spectra, absorption spectra, melting temperature, ionic strength effect, and viscosity experiments were described that characterize the interaction of eugenol with salmon sperm DNA in vitro. Eugenol was found to bind but weakly to DNA, with binding constants of 4.23×10³, 3.62×10³ and 2.47×10³ L mol^?1 at 18, 28 and 38 °C respectively. The Stern–Volmer plots at different temperatures suggested that the quenching type of fluorescence of eugenol by DNA was a static quenching. Both the relative viscosity and the melting temperature of DNA were increased by the addition of eugenol. The changes of ionic strength had no affect on the binding. In addition, the binding constant of eugenol with single stranded DNA (ssDNA) was larger than that of eugenol with double stranded DNA (dsDNA). These results revealed that the binding mode of eugenol to DNA was intercalative binding. The thermodynamic parameters ΔH, ΔG and ΔS were also obtained according to the Van't Hoff equations, which suggested that hydrogen bond or van der Waals force might play an important role in a binding of eugenol to DNA. Based on the theory of the Förster energy transference, the binding distance between DNA and eugenol was determined as 4.40 nm, indicating that the static fluorescence quenching of eugenol by DNA was also a non-radiation energy transfer process.     

Synthesis, Spectroscopy and Photochemistry of Nitro-Azobenzene Dyes Bearing Benzophenone Parts

Novel nitro-azobenzene dyes bearing one or two benzophenone branches were proposed and synthesized to improve their photophysical and photochemical properties. The new dyes exhibited double UV/visible bands, and they displayed weak fluorescence emission as excited at 350 nm. Single crystal X-ray diffraction data showed that two phenyl rings of azobenzene was almost coplanar, and the benzophenone part was neither coplanar nor linear connection with azobenzene via ether bridged bond, which have good fit with molecular geometry optimization calculation results. The cyclic voltammeric results of nitro-azobenzene dyes were firstly reported in this paper, which demonstrated that the electrochemical properties of nitro-azobenzene dyes was altered by the substitution of benzophenone part. Thermal stabilities of the new dyes were studied by the analysis of differential scanning calorimetry (DSC) and thermograving (TG) in this paper. Efficient visible-light photoinitiating polymerization of methyl methacrylate (MMA) by the novel nitro-azobenzene dyes was presented and discussed.     

Surface modification of the polyethyleneimine layer on silicone oxide film via UV radiation

We herein report a novel method of employing 254 nm of UV radiation (UV) for the modification of a polyethyleneimine (PEI) layer on silicone oxide film. In this study, a PEI layer composed of a 50 mM sodium carbonate solution (pH 8.2) was formed on the surface of a silicone oxide film with spontaneous adsorption. Then, thin film of PEI was patterned by UV radiation. To determine the effect of the UV radiation, fluorescence microscopy, X-ray electron spectroscopy (XPS), and Fourier Transform Infrared spectroscopy (FT-IR) analyses were performed. These results indicated that UV radiation could cause changes in the surface characteristics of the PEI layer. Subsequently, FT-IR analysis showed changes in the chemical composition of the PEI exposed to UV radiation, such as the disappearance of the amine. Based on these results, we can conclude that UV radiation could be used to eliminate the amine group selectively and that this technique could be applied to create a pattern on the surface of a PEI layer.     

Effects of different levels of vitamin C on UV radiation-induced DNA damage

The Raman spectra of DNA in different levels of vitamin C with 10- and 30-min ultraviolet (UV) radiations were reported. The intensity of UV radiation was 18.68 W/m2. The experimental results proved that vitamin C could alone prevent UV radiation from damaging DNA, but the effects depended on the concentration of vitamin C. When the concentration of vitamin C was about 0.08-0.4 mmol/L, vitamin C decreased UV radiation-induced DNA's damage. When the concentration of vitamin C exceeded 0.4 mmol/L, vitamin C accelerated DNA's damage instead. Maybe the reason is that when DNA in aqueous solution is radiated by UV, free radicals come into being, and vitamin C can scavenge free radicals, so vitamin C in lower concentration can protect DNA. The quantity of free radicals is finite, when vitamin C is superfluous, free radicals have been scavenged absolutely and vitamin C is residual. Vitamin C is a strong reductant. When the mixture of DNA and residual vitamin C is radiated by UV, vitamin C reacts with DNA. The more residual vitamin C and the longer time of UV radiation, the more DNA is damaged.